Inward Rectifyimg Potassium Channel ---The Experiment Investigation That Puerarin Prevents And Treats Arrhythmia And It's Mechanism

Background and objectiveSevere arrhythmia is one of the main death causes from cardiacdisease. Although the method of treating arrhythmia that clinical electrophysiologic study (EPS) and radio-frequency ablation is done by transcather gains tremendous success, the druges preventing and treating arrhythmia still are one of main means that reduce the rate of death and improve patient's quality of life. At present, anti-arrhythmia medicines act on mainly Na+, Kv and Ca2+ ion channel. The medicines are the doctor's magic weapons of anti-arrhythmia. But their pro-arrhythmia effect causes widespread worry. Developing newanti-arrhythmia druge has become the medical worker's general view. The relation between inward rectifier potassium channel and anti-arrhythmia is unclear. Puerarin' effect on myocytes ion channel has not reported yet.In order to find if the druges act on Iki to produce antiarrhythmia, present experiment investigated the electrophysiological nature of Ikiand the effects of Puerarin on ion channels and arrhythmia in organ and whole-life level model. Methods and resultPART I To obtain the mRNA of Iki by in vitro transcription method of cDNA, to inject the mRNA into Xenopus oocytes by nano-injection technique for heteroexpression and to culture oocytes; to record Iki channel current by two-electrode voltage clamping to study the electrophysiological properties and the action of Puerarin and Ang II-Antipeptide in different concentrations including extracellular [K+] dependence; to analyze the electrophysiological data by pCLAMP and Origin 6.0. Result: 1 .Affirming transcription product mRNA is Iki by electrophoresis technology. 2. Ikl possesed distinct inward rectifier property at membrane hyperpolarization. Outward current is apparent at membrance potential from ?0 mV to ?0 mV; Ba2+(metal cation) can blocked Iki selectively. 3. Puerarin inhibited the inward and outward currents of Ik]. Inhibition increases with the enhancement of druge concentration; Puerarin' inhibition reinforce when perfusing higher potassium solution for the oocytes. 4. Effect of Puerarin on Ikl was dose-dependent. It was reversible. 5. Ang II-Antipeptide didn't effect onlki.PART II Ionic channel currents of isolated adult rat ventricular myocytes were investigated by using whole-cell path-clamp technique. Result: 1. Puerarin decreased Iki instantanoues current and steady state current elicited by 500 mes duration of pulses depolarized to different membrance potential, and the blockade was dose-dependent. E-4031 didn't effect on Ikl. 2. There was not distinct inhibition to Ito with low-concentration puerarin (1.2~9.6mmol/L), but high-concentration puerarin (19.2mmol/L) was efficiency on Ito (vs contrast group 21 ?6.3%, n=5, p<0.05). Low-concentration E-4031 (1.2~9.6umol/L) didn't effect on Ito but high-concentration E-4031 (19.2u.mol/L) was efficiency on Ito (19 + 4.1%, n=5, p<0.05 vs contrast group peak current). The inhibition of puerarin to Iki was stronger than one for Ito. 4. Puerarin and E4031 had little effects on INa and IcaL.PART III Building arrhythmia models of heart produced by aconitine in vitro. The rats were assigned into treating arrhythmia suit and preventing arrhythmia suit. Result: (1) Inhibit rates of O.lmg, 0.2mg and 0.3mg Puerarin on arrhythmia induced o.lmmol/L aconitine 0.7ml were 70%, 100% and 100%. There were not changes of LVT, dp/dt/mmHg/s and HR. (2) Injecting E-4031 (3ug) can't inhibit arrhythmia. (3)Iinjecting 0.2mg puerarin, aconitine as same dose above can't induce arrhythmia. (4) After injecting 0.1 mg, 0.2mg and 0.3mg puerarin, the doses of aconitine (pouring O.ljimol /0.2jig/min) producing arrhythmia had significantly increased (n=30, P<0.01). ii. Building arrhythmia models of heart produced by aconitine in vivo. Groups were divided as the experiment of vitro. Result: (1) 130mg/kg, 200mg/kg and 270mg/kg puerarin can inhibit the arrhythmia by 40u,g/kg aconitine. Inhibit rates were 80%, 100% and 100%(n=30, p<0.01). There were not changes of LVT, dp/ dt/ mmHg/s and HR i...